Journal of Molecular Liquids 377 (2023) 121511 Contents lists available at ScienceDirect Journal of Molecular Liquids journal homepage: www.elsevier.com/locate/molliq Light excitation of gold Nanorod-Based hybrid nanoplatforms for simultaneous bimodal phototherapy Lilia G. Arellano a,1, Eva M. Villar-Alvarez b,1, Brenda Velasco a, Vicente Domínguez-Arca a, Gerardo Prieto c, Adriana Cambón a,⇑, Silvia Barbosa a,⇑, Pablo Taboada a a Grupo de Física de Coloides y Polímeros, Departamento de Física de Partículas; Facultad de Física e Instituto de Materiales (iMATUS) e Instituto de Investigaciones Sanitarias (IDIS), Universidade de Santiago de Compostela, 15782 Santiago de Compostela, Spain b Nanostructured Functional Materials Group, Catalan Institute of Nanoscience and Nanotechnology, UAB Campus, 08193 Bellaterra (Barcelona), Spain c Grupo de Biofísica e Interfases, Departamento de Física Aplicada, Facultad de Física e Instituto de Materiales (iMATUS), Universidad de Santiago de Compostela, 15782 Santiago de Compostela, Spain a r t i c l e i n f o a b s t r a c t Article history: In this work, we developed a hybrid nanosystem able to simultaneously combine its potential as a pho- Received 5 December 2022 todynamic (PDT) and plasmonic photothermal (PPTT) therapeutic agent to kill malignant cells. To do that, Revised 8 February 2023 AuNRs were functionalized by means of the layer-by-layer (LbL) assembly technique using alternating Accepted 17 February 2023 layers of anionic poly(styrene sulfonate) (PSS) and cationic poly-L-lysine (PLL) as polyelectrolytes Available online 24 February 2023 (PEs), and an outer final layer of hyaluronic acid (HA) to provide the hybrid particles with both sufficient colloidal stability and targeting ability to tumoral cells overexpressing CD44 receptors. To provide the Keywords: nanoplatform with PDT capabilities, the near-infrared (NIR) sensitive photosensitizer (PS) indocyanine Gold nanorods Layer by layer green (ICG) was previously grafted to the PLL PE and assembled on the particle surface coating; in this Photodynamic therapy manner, PSS/PLL-ICG/HA-coated AuNRs hybrid particles were obtained. Then, the conditions for opti- Isocyanine gree mized reactive oxygen species (ROS) production under NIR light excitation were elucidated, reaching Phothermal therapy ca. 80% after 5 min irradiation at 2.0 W/cm2 compared to positive control (H2O2). Since ICG can also absorb near infrared (NIR) light and transforms it by internal conversion into heat under suitable irradi- ation conditions, which adds to the photothermal plasmonic effect provided by the metallic NP, the heat- ing profiles provided by the present hybrid nanoparticles (NPs) were measured, being observed temperature increments ranging from 9 to 22 °C. The potential contribution of its different components to both PDT and PPTT were deeply analyzed in vitro for intended cancer therapeutics at several power intensities but also at different temperatures with the aim of elucidating the role played by both pho- totherapies on cell cytotoxicity, the optimal illumination conditions for effective bimodal phototherapy as well as the elucidation of the main cell death mechanism involved. Under optimized conditions and after the administration of 2.5 1010 NPs/mL, cell cytotoxicites of up to ca. 70% were determined by com- bination of PDT + PPTT therapeutic effect. Ó 2023 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). 1. Introduction kill them (thermal ablation regime, T > 45 °C) or to sensitize them (thermal sensitization, T < 45 °C) in order to either enhance/com- Plasmonic photothermal therapy (PPTT) is a newly emerging plement the therapeutic outcome of an additional first line applied tool to fight against different diseases as cancer which makes use therapy (i.e., radio- or chemotherapy), and/or to induce a suitable of the outstanding optical properties of plasmonic nanoparticles cytostatic pathway leading to cell death (i.e., apoptosis). At this (NPs). Briefly, PPTT is a minimally-invasive therapeutic strategy respect, Au NPs (i.e., nanospheres, nanorods, nanoshells, nanocages in which photon energy is transformed into localized heat leading and nanostars) have attracted great attention as potential pho- to sufficient temperature (T) increments in tumoral cells to directly tothermal agents [1,2] thanks to their high-absorption cross sec- tions for conversion of light into heat. In addition, Au NPs are also known to be biocompatible and, hence, they can be served ⇑ Corresponding authors. as nanocarriers for drugs and other bioactive molecules; and their E-mail addresses: adriana.cambon.freire@usc.es (A. Cambón), silvia.barbo- sa@usc.es (S. Barbosa). easy surface chemistry allows the attachment of new functionali- 1 These authors contribute equally to this work. ties as ligands (antibodies, peptides, proteins, etc) for targeted https://doi.org/10.1016/j.molliq.2023.121511 0167-7322/Ó 2023 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). L.G. Arellano, E.M. Villar-Alvarez, B. Velasco et al. Journal of Molecular Liquids 377 (2023) 121511 cargo delivery and cell uptake and internalization into tumoral electric charge. A hyaluronic acid (HA) outer coating layer was cells [3,4]. deposited onto the NPs to provide the hybrid nanoplatform with Amongst the different types of metallic NPs, gold nanorods active targeting capabilities by exploiting the high affinity of this (AuNRs) are particularly appealing thanks to their anisotropic biopolymer to bind CD44 receptors overexpressed on the mem- structure which allows the electronic oscillations to occur mainly brane surfaces of different tumors as breast, lung and colorectal along the short and long axes of the particle resulting in the gener- cancers [29,30]. To provide the platform with PDT capability, the ation of transverse and longitudinal localized surface plasmon indocyanine green (ICG) dye was also assembled onto the AuNR- bands (LPSRs) [5,6]. The transversal band is regularly observed in surface coating. ICG is a tricarbocyanine compound with absorbing the visible region of the electromagnetic spectrum, whilst the lon- properties in the NIR approved by the US Food and Drug Adminis- gitudinal one is displayed in the near infrared region (NIR) of the tration (FDA) for medical diagnostics as a contrast agent for blood electromagnetic spectrum, which can be easily tuned by modifying volume determination, ophthalmic angiography, cardiac output, the AuNR aspect ratio. AuNRs exhibit the largest NIR absorption and hepatic function [31,32] but also a potential PDT agent for can- cross-section compared to other Au NPs [7], demonstrating an cer treatment [33,34]. As a consequence of the interaction between extremely efficient NIR photothermal heat conversion [8,9]. Fur- metallic nanomaterials and the excited state of fluorophores, free thermore, AuNRs possess much larger surface areas per volume electrons in metals can oscillate with those of fluorophores to pro- than spherical Au NPs, making them an attractive potential duce a dipolar interaction which may result in an enhancement of nanocarrier with large cargo loading capability, especially useful light absorption, emission, and/or stability of the fluorophore to co-deliver different types of therapeutical agents to cancer cells excited state to provide more efficient single PDT and/or combined [10,11]. PPTT and PDT [35–39]. Nevertheless, poor attention has been paid While these plasmonic nanostructures can certainly increase to both identify suitable irradiation conditions for effective either the local temperature in cancerous areas upon exposition to NIR single or dual PDT-PPTT applied therapies for synergistic therapeu- light of suitable wavelengths, it is not completely evident that tic outcomes using multimodal nanoplatforms, and to decipher the the resulting photothermal effect is enough to achieve the com- predominant cell death mechanism involved in each case. Hence, in plete eradication of malignant cells while avoiding damage of the this work we also focused on the analysis of the photostability of surrounding healthy tissue [12,13]. Thus, the development of the encapsulated ICG PS inside the polymeric coating of the new nanostructures able to enhance cancer treatment efficacy by nanoplatform, as well as in the role played by illumination condi- the simultaneous combination of different phototherapeutic tions on both the NIR-induced temperature heat profiles and ROS approaches is vital and can allow to overcome current limitations production inside tumoral cells. The developed hybrid nanosystem of single therapies while leading, at the same time, to potential was observed to be an ideally versatile platform to simultaneously synergistic cytotoxic effects [14–16]. deliver localized heat and ROS, whilst providing an efficient protec- Photodynamic therapy (PDT) is a treatment modality for cancer tion of the ICG dye molecules to avoid their early clearance from the and other diseases in which targeted cells are selectively destroyed body. Thus, combined PDT and PPTT may kill cancer cells more effi- by the biological activity of light-sensitive compounds known as ciently than PDT or hyperthermia treatments alone whereas the photosensitizers (PSs) [17,18]. When exposed to light of an appro- predominance of a single or dual therapeutic mode was dependent priate wavelength, these PSs become energized and generate reac- on illumination and medium conditions, which was reflected both tive oxygen species (ROS) including singlet oxygen (1O2) to kill in the cytotoxic response provided as well as in the cell death mech- cancer cells [18–20]. ROS are naturally generated as a result of anisms involved. the reduction of oxygen during aerobic respiration and by various enzymes within cells. At physiological levels, ROS contribute to cell 2. Materials and methods signaling and host defense mechanisms [21]. Enhanced ROS levels, above the detoxification capacity of a biological system, may result 2.1. Materials. in significant damage of cell structures and oxidative stress, partic- ularly, by means of the alteration of biocromolecules such as Hexadecyltrimethyl ammonium bromide for molecular biology polyunsaturated fatty acids in lipid membranes, essential proteins (CTAB), tetrachloroauric acid (HAuCl43H2O), silver nitrate and DNA giving rise to, for example, cell apoptosis [22]. Thus, (AgNO3), sodium borohydride (NaBH4), poly(sodium-4- tumors might be then selectively destroyed by PDT using localized, styrenesulfonate) (PSS) of molecular weight (Mw)  70000 g/mol, light-excited PSs avoiding the severe side effects associated with poly-L-lysine hydrobromide (PLL) of Mw  22000 g/mol, hyaluro- other systemic conventional treatments such as chemotherapy; nic acid of Mw  15000 g/mol,1,3-diphenylisobenzofuran (DBIPF) but also PDT is able re-sensitize cancer cells to chemotherapy and indocyanine green (ICG) were purchased from Sigma-Aldrich through intrinsic regulation of intracellular redox systems, cell (USA). ICG sulfo-NHS ester was from BioActs (Korea). Ascorbic acid death mediators as well as effective activation of immune cells. was from Fluka. Heat- inactivated fetal bovine serum (FBS), However, most currently used PSs possess several important disad- trypsin-EDTA (0.25X) and PBS pH 7.4 (10X) were from Hyclone vantages as their hydrophobic nature, short retention times, and/or (Thermo Scientific, USA). Prolong Antifade reagent with DAPI was poor selectivity to diseased cells and tissues, amongst others. Thus, from Molecular Probes (USA), and the Cell Death Annexin V - Pro- increasing research efforts are being devoted for developing new pidium Iodide kit was from Gerbu Biotechnik GmbH (Germany). All potential PSs to overcome the former limitations. other reagents were of analytical grade and/or suitable for cell cul- Therefore, PPTT and PDT appear as two photon-mediated thera- ture as corresponding. All chemicals were used as received. Milli-Q peutic methods that might be combined in one single nanoplatform water (Millipore) was used throughout all the experiments. for efficient multimodal cancer therapy to achieve simultaneous ROS and hyperthermia-mediated cellular damage [15,23–28]. Hence, with this aim in mind, in this work we developed a hybrid 2.2. Synthesis of AuNRs. nanoplatform able to simultaneously combine PDT and PPTT ther- apeutic activities to treat malignant cancer cells. This nanocon- AuNRs were synthesized using a seed- mediated growth struct was based on surface-functionalized AuNRs by means of method. First, CTAB-capped Au seeds were obtained. To do that, the layer-by-layer (LbL) technique using poly(styrene sulfonate), 7.5 mL of a 0.2 M CTAB solution was gently mixed with 0.25 mL PSS, and poly(L-lysine), PLL, as polymeric coating layers of opposite of 0.01 M HAuCl4 in a water bath at 28 °C. As indication, for both 2 L.G. Arellano, E.M. Villar-Alvarez, B. Velasco et al. Journal of Molecular Liquids 377 (2023) 121511 seeds and AuNRs preparation, CTAB was left at a constant temper- AuNRs were centrifuged at 14,000 rcf for 10 min and redispersed in ature of 28 °C for one day under constant stirring of 200 rpm to 1 mL of MilliQ water. achieve full solubilization and avoid foaming prior to use. Next, a 0.01 M NaBH4 solution was prepared in ice-cold water. This solu- 2.4. Nanoplatform characterization tion was left to rest for 2–3 min to ensure a good dispersion of the reductant. Afterwards, while the Au-CTAB solution was stirred 2.4.1. Dynamic light scattering (DLS). at 200 rpm, 0.6 mL of ice-cold 0.01 M NaBH4 were added in one DLS measurements were done using an ALV-5000 digital corre- pull to the former, after which the mixed solution turned brownish lator system (ALV 5000/E, ALV GmbH, Germany) equipped with a yellow. This solution was mixed gently by hand for 2 min and then temperature control set at 25 °C ± 0.1 °C. The scattered light was left undisturbed in a water bath at 28 °C for 1 h to allow the excess vertically polarized with a 532 nm solid-state laser (2 W). The sodium borohydride to be decomposed. hydrodynamic radius, Rh, was obtained for diluted samples from For AuNRs growth, 850 lL of a 0.01 M HAuCl4 solution were DLS measurements at an incidence angle of 90° by analysis of the added to 20 mL of 0.2 M CTAB in a water bath at 28 °C, after which DLS data by means of the CONTIN algorithm [40] and the Stokes- the solution turned yellow bright while stirred at 500 rpm. Then, Einstein equation, Rh = kBT/6pgD, where kB is the Boltzmann con- 0.01 M of an AgNO3 solution was prepared in darkness. Different stant, T the temperature, g the solution viscosity, and D the diffu- volumes of the silver solution (ranging from 126 lL to 246 lL) sion coefficient of the particles. Measurements were performed at were added to the Au growth solution followed by gentle mixing least in triplicate with a sampling time of 60 s each and averaged. at 500 rpm. Then, 136 lL of a 0.1 M ascorbic acid (AA) solution were added followed by gentle stirring at 500 rpm until the solu- 2.4.2. Inductively coupled plasma-mass spectrometry (ICP-MS). tion turned colourless (ca. 4 min). Finally, 220 lL of the Au seed The Au concentration in solution or in cells was determined by solution were gently added to the Au growth solution while stir- inductively coupling plasma mass spectrometry (ICP-MS) in a Var- ring for 2 min and, then, stopped. The resulting solution was left ian 820-MS equipment (Agilent Technologies, USA). 1 mL of AuNRs undisturbed in a water bath overnight at 28 °C for ca. 12 h becom- solution (or 100,000 cells with internalized metallic NPs, where ing reddish-pink. The formed AuNRs were centrifuged at least corresponding) was dissolved in 0.3 mL HCl 37% (v/v) and 0.1 mL twice at 28 °C for 30 min at ca. 29,500 rcf and redispersed in HNO3 70% (v/v). Solutions were diluted with deionized water until 20 mL of deionized water. The UV– visible absorption spectra of reaching a final volume of 2 mL. The intensity of the emission the obtained AuNRs were measured using a Cary Bio 100 UV–vis wavelength was measured and compared to a standard solution. spectrophotometer (Agilent Technologies, USA). The sizes of the AuNRs were measured using a JEOL JEM 1011 (Japan) transmission 2.4.3. Electrophoretic mobilities. electron microscope operating at an accelerating voltage of 120 Potentials of bare and PE-coated AuNRs were measured using a kVand aspect ratios (ARs) calculated. Nano ZS instrument (Nanoseries, Malvern Instruments, UK) The concentration of AuNRs per mL was calculated by means of equipped with a 633 nm He-Ne laser of 4 mW. The equipment inductively coupled plasma mass spectroscopy-MS (ICP-MS). For measures the electrophoretic mobility of the particles and converts example, for the case of AuNRs with a longitudinal LSPR band cen- it into fpotential data using the classical Smoluchowski equation: tered at 792 nm, 1 mL of three different AuNRs batches at an OD  1 has an Au concentration of ca. 49.2 ± 0.4 lg/mL taking into a ¼ ef=g ð1Þ account the particle length and width obtained by TEM. The final where a, e, f, and g denote the electrophoretic mobility, per- concentration obtained under these conditions was of ca. 51011- mittivity of the medium, the fpotential of the particles and med- AuNRs/mL. ium viscosity, respectively. Each sample was fed into a folded capillary, clear, disposable cell. Measurements were initiated after 2.3. LbL polymeric coating of AuNRs. attaining thermal equilibrium at 25 °C. The number of runs for each experimental point was automatically determined by the Using the LbL methodology, multilayers of PSS, PLL and HA were software, and measurements were performed in triplicate. Results successfully deposited onto the AuNR surfaces. For the PSS coating, were reported as the mean ± standard deviation (SD). Solution with a solution of PSS (10 mg/mL) in 12 mM NaCl (100 mL) was pre- concentrations lower than 100 lgmL1 were used. pared. When this PE was completely dissolved, 1 mL of this solu- tion was mixed with 1 mL of a 12 mM NaCl solution, and the 2.4.4. Encapsulation efficiency (EE%) of ICG inside the hybrid resulting mixture stirred at 500 rpm. Then, 1 mL of an AuNR solu- nanoplatform: tion (OD  1) was added dropwise in the PSS solution while stir- To determine the EE% of ICG within PSS/PLL-ICG/HA-coated ring. After 1 h of adsorption, the mixture was centrifuged twice AuNRs, the hybrid NPs were centrifuged at 15000 rpm at 20 °C for at 31,500 rcf for 20 min and resuspended in 1 mL of MilliQ water. 20 min. The ICG content in the supernatant was measured by means Next, a subsequent cationic PLL-ICG layer can be used to coat the of fluorescent spectroscopy using a Cary Eclipse spectrophotometer hybrids. To do that, a volume of 100 mL of PLL (5 mg/mL) was pre- (Agilent Technologies, USA). Previously, a fluorescence calibration viously well mixed with a solution containing ICG-sulfo-NHS ester curve with free ICG was obtained using light with kex = 785 nm (0.02 mg of ICG, 0.02 mg/mL). The mixture was allowed to react for and kem = 810 nm, and the fluorescence of solution supernatants 2 h at room temperature in the dark, obtaining a PLL-ICG complex of bare AuNR solutions were also considered as an additional blank. by carbodiimide chemistry. Next, to remove unbound ICG, the mix- Each sample was measured in triplicate for three different batches. ture was dialyzed for 24 h against water with a cellulose dialysis The EE% was calculated by the following expression: membrane with MWCO of 3500 Da. Then, PSS-coated AuNRs were added dropwise under stirring at 500 rpm. After 1 h of adsorption, Total amount of dye feeded  dye in supernatant EEð%Þ ¼  100 the coated metallic NPs were centrifuged at 31,500 rcf for 20 min Total amount of dye feeded and resuspended in 1 mL of MilliQ water. The deposition of the ð2Þ final HA layer was made by preparing 60 lL of a HA concentrated solution (1 mg/mL), mixed with 1 mL of MilliQ water and stirred at 2.4.5. Photostability of ICG assembled onto AuNRs: 500 rpm for 5 min. 1 mL of the former PSS/PLL-ICG-coated AuNRs The stability of free ICG and PSS/PLL-ICG/HA-coated AuNRs was added dropwise to the HA solution. After 1 h, the final coated (1 mL, OD  1, ca. 11011AuNRs/mL, 22 lM of dye) in water was fol- 3 L.G. Arellano, E.M. Villar-Alvarez, B. Velasco et al. Journal of Molecular Liquids 377 (2023) 121511 lowed for ca. 20 days by measuring their fluorescence decrease. buffer to maintain the required sink conditions. The ICG content Fluorescence spectra were monitored in a Cary Eclipse spectropho- in the supernatant was measured by means of fluorescence spec- tometer (Agilent Technologies, USA). The excitation was set at troscopy using a previously established calibration curve in the kex = 785 nm and the emission peak recorded at kem = 810 nm. Also, corresponding buffer solutions. Fluorescence spectra were moni- the stability under NIR light irradiation was analyzed at several tored in a Cary Eclipse spectrophotometer (Agilent Technologies, light fluencies (0.5, 1.0, 2.0 W/cm2). The samples were magneti- USA). Assays were carried out in triplicate. cally stirred and illuminated with NIR-light (808 nm) for a total time of 20 min. The fluorescence spectra were recorded at several 2.7.1. Protease-assisted release. times and every experimental point repeated at least three times. To measure the enzymatic release of ICG from AuNRs, 1 mL (1.01011AuNRs/mL) was incubated into dialysis tubes (Spec- 2.5. NIR-laser induced photothermal effect of AuNRs. traPoreÒ, MWCO 3500 Da) at 37 °C under moderate stirring (300 rpm) for several days at pH 7.4 and 5.0 in the absence or pres- Temperature increment tests were performed using a continu- ence of trypsin–EDTA (50 lL per 25 mL of buffer) and immersed ous wave fiber-coupled diode laser source of 808 nm wavelength into 25 mL of buffer supplemented with 10% (v/v) FBS. The released (50 W, Oclaro Inc., San Jose, CA). The laser was powered by a New- ICG concentrations were determined as explained above. port 5700–80 regulated laser diode driver (Newport Corporation, Irvine, CA). A 200-lm-core optical fiber was used to transfer the 2.7.2. NIR-light triggered release. laser light from the laser unit to the target solution and it addition- To analyze the effect of laser exposure on ICG release from ally was equipped with a lens telescope mounting accessory at the AuNRs, 1 mL (1.01011 NP/mL), the present hybrid NPs were incu- output, which allowed the fine tuning of the laser spot size in the bated at 37 °C under moderate stirring (300 rpm) at pH 5.0 inside range of 0.1–10 mm. The laser spot size was measured with a laser the dialysis bags as previously commented. The experimental con- beam profiler (Newport LBP-1-USB) placed at the same distance ditions were similar as those previously stated except that AuNRs (8 cm) between the lens telescope output and the 3 mL cuvette were exposed to NIR light irradiation of 1.0 and 2.0 W/cm2 for (or the 6-well plate when corresponding), using the software New- 5 min after 2, 5, 9, 12 and 48 h of incubation. Irradiation was per- port LBP series Measurement Systems v3.11. In this way, the power formed as described above for photothermal experiments. per unit area was easily obtained. The final spot size was set at 1 cm in diameter. A potentiometer (Newport Optical Power Meter 2.8. Cell cultures. model 1916c) was used to calibrate the output power related to the intensity signal of the laser controller. The temperature of Cervical HeLa cancer cells and 3 T3 mouse fibroblasts from Cell AuNRs samples was measured with a type J thermocouple linked Biolabs (San Diego, CA) were grown at standard culture conditions to a digital thermometer inserted into the solutions. Particle solu- (5% CO2 at 37 °C) in DMEM supplemented with 10% (v/v) FBS, tions were stirred during laser illumination to homogenize the pro- 2 mM L-glutamine, 1% (v/v) penicillin/streptomycin, 1 mM sodium duced heat and ensure that samples were in thermal equilibrium pyruvate, and 0.1 mM MEM non-essential amino acids (NEAA). during the entire course of the experiments. 2.9. Cellular uptake by fluorescence microscopy. 2.6. In vitro ROS generation. Cervical HeLa cancer cells were cultured in poly-L-lysine-coated The in vitro generation of ROS was performed by following the glass coverslips (12  12 mm2) placed inside 6-well plates (2 mL, absorbance decrease at 420 nm of 1,3-diphenylisobenzofurane 1.0105 cells per well) and grown for 24 hat standard culture con- (DPIBF), which is a specific singlet oxygen trap [41]. Briefly, 1 mL ditions. Then, the culture medium was discarded. 100 lL of PSS/ of the corresponding sample (deionized water, free ICG, PSS/PLL/ PLL-ICG/HA-coated AuNRs (2.51010AuNRs/mL) were redispersed HA-coated AuNRs and PSS/PLL-ICG/HA-coated AuNRs) were placed in cell culture medium with 10% (v/v) FBS and added. After 6 h in a quartz cell, and 100 lL of 1 mM DPIBF in ethanol were added. of incubation, the NP containing cells were washed three times The concentration of free ICG and ICG bound to PSS/PLL-ICG/HA- with PBS pH 7.4, and then fixed with paraformaldehyde 4% (w/v) coated AuNRs was ca. 22 lM (1 mL, OD  1, ca. 11011AuNRs/ for 10 min, washed with PBS, permeabilized with 0.2% (w/v) Triton mL). UV–vis spectra of samples before illumination were recorded X-100, and stained with BODIPY Phalloidin. Cells were washed and taken as the initial (100%) absorbance value. The sample was again with PBS, mounted on glass slides stained with DAPI, and magnetically stirred and illuminated with NIR light using a CW- cured for 24 h at 20 °C. Samples were visualized with a 63X 808 nm laser source at different time points. Then, the UV–vis (oil-immersion, NA 1.4) objective using a confocal spectral micro- spectra were recorded and the process was repeated twice more. scope Leica TCS-SP2 (LEICA Microsystems Heidelberg GmbH, Man- The ROS generation was qualitatively determined at three different nheim, Germany), whereby the blue channel corresponds to DAPI laser fluencies: 0.5, 1.0, and 2.0 W/cm2. (kex = 355 nm), the pink channel to BODIPY Phalloidin (kex = 633 nm) and transmitted light to differential interference contrast (DIC)- 2.7. In vitro ICG release. mode. Green channel corresponds to the reflected light emitted by the nanoplatform. Additional stability studies were performed by analyzing the in vitro ICG release profiles from the obtained hybrid nanoplat- 2.10. Fluorescence microscopy ROS assay in tumoral cells. forms at a constant temperature of 37 °C and 300 rpm magnetic stirring for several days. PSS/PLL-ICG/HA-coated AuNRs were incu- A fluorometric intracellular ROS kit (Sigma-Aldrich, USA) was bated at pH 7.4 and pH 5.0 (1 mL, 11011AuNRs/mL). To obtain the used to detect ROS according to manufacturer’s instruction. Briefly, release profiles, 1 mL of hybrid AuNRs was placed into dialysis 1.0105 HeLa cells/well were seeded on 6-wellplates with 2 mL of tubes (SpectraPoreÒ, MWCO 3500 Da) immersed into 50 mL of buf- DMEM without red phenol and grown for 24 h at standard culture fer supplemented with 10% (v/v) FBS at the pH of interest. The conditions. Afterwards, 500 lL of PSS/PLL-ICG/HA-coated AuNRs at released ICG concentration was determined at different time inter- a concentration of 2.51010AuNRs/mL were added to the wells and vals for each pH solution. At each sampling time, 1 mL of the med- incubated for 6 h. Then, the medium was discarded, cells were ium was withdrawn and replaced with the same volume of fresh washed three times with PBS and, finally, fresh culture medium 4 L.G. Arellano, E.M. Villar-Alvarez, B. Velasco et al. Journal of Molecular Liquids 377 (2023) 121511 without red phenol was added. Then, 100 lL of ROS detection plasmon band at ca. 512 nm and a stronger longitudinal one at ca. reagent were added to each well and cells were incubated for 1 h 792 nm, respectively. To mask the remaining CTAB molecules at 37 °C. Next, cells were treated with a CW diode laser (808 nm) adhered to the NPs surface after the synthesis and subsequent at 1.0 W/cm2 for 5, 10 or 15 min. As a negative control some cells washing steps, which are potentially toxic to cells and tissues were left untreated (without NPs), and as a positive control 100% and provide the metallic particles with colloidal stability, particleś ROS was induced by adding in this step H2O2 (800 lM, 33% (w/ surfaces were modified through the layer-by-layer (LbL) self- v)). Then, cells were left in the incubator other 30 min. Samples assembly technique. Hence, sequential layers of anionic PSS, catio- with live cells were visualized at 20X using an inverted wide field nic ICG-modified PLL, and anionic HA polymers were alternatively and fluorescent microscopy (Leica DMI6000B, Leica Microsystems, deposited onto the surface of NPs to give PSS/PLL-ICG/HA-coated Germany) using the green channel for ROS fluorescence signal AuNRs. The outer HA layer was incorporated to reduce the total (kex = 490 nm/kem = 520 nm), and the transmitted light in differen- positive surface electrical charge of the nanoplatform in order to tial interference contrast (DIC) mode. avoid potential cell toxic effects due to excessive surface positive electric charge as well as allowing specific targeting to CD44 recep- 2.11. In vitro cell cytotoxicity. tors commonly overexpressed in the cytoplasmatic membrane of several types of malignant stem and cancerous cells, as previously Cytotoxicity of PSS/PLL-ICG/HA-coated AuNRs was tested mentioned [42,43]. To provide the nanoplatform with PDT and in vitro by means of the CCK-8 cytotoxicity assay. Cancerous cervi- optical imaging capabilities, the cationic PLL polymer was conju- cal HeLa and 3 T3 Balb fibroblasts cells were seeded into 96-well gated with ICG molecules by carbodiimide chemistry. The effi- plates (1.0104 cells/well) and grown for 24 h at an optical conflu- ciency of ICG complexation with PLL was ca. 94% and the degree ence of 80–90% under standard culture conditions in 100 lL of substituted PLL amino groups with ICG was 0.1%, which does growth medium. PSS/PLL/HA-coated AuNRs, PSS/PLL-ICG/HA- not interfere with the cationic charge of the PE in the LbL self- coated AuNRs and free ICG were used as negative and positive con- assembly procedure. trols, respectively. After 24 h of incubation at 37 °C, 100 lL of NPs Fig. 1a-b shows the main physico-chemical characteristics of at 2.51010 NP/mL in the corresponding cell culture medium were the obtained PSS/PLL-ICG/HA-coated AuNRs. The transverse plas- injected into the wells and incubated for 24 h and 48 h. Some wells mon peak located at ca. 512 nm almost remained invariable as were tested in the absence of NPs as a negative control (blank), and the consecutive layers containing PSS, PLL-ICG and HA were depos- with free ICG at the same concentration as that encapsulated in the ited onto the metallic NPs. In contrast, the longitudinal LSPR band hybrid AuNR nanoplatform. After incubation, the culture medium changed upon PE deposition as a consequence of the sensitivity of was discarded, cells were washed with 10 mM PBS (pH 7.4) several the metallic NPs to the dielectric properties of the surrounding times, and fresh culture medium (100 lL) containing 10 lL of CCK- environment [44,45]. This confirmed the successful surface poly- 8 reagent added to each well. After 2 h, the absorption at 450 nm of meric coating. The initial blue-shift of the LPSR band after PSS cell samples was measured with an UV–vis microplate absorbance deposition may be related to the lower refractive index (RI) of this reader (Bio-Rad model 689, USA). Cell viability (SR, survival rate) PE compared to that of CTAB (1.43 vs. 1.38) [46]. Conversely, the was calculated as follows: opposite trend, that is, a red shift of the LPSR maximum, was noted when adsorbing the PLL-ICG polymer conjugate (RI of PLL = 1.52). A Abssample certain broadening of the LPSR peak after the final HA layer was SR ¼  100 ð3Þ Absblank observed which is probably related to the formation of some par- where Abssample is the absorbance at 450 nm for cell samples, ticle clusters, as seen in TEM images (Fig. 1f-g). The successful presence of the ICG dye within the particle coating was addition- and Absblank is the absorbance corresponding to the sample con- trols without the particles. In addition, some of the wells were also ally confirmed by the development of a shoulder at ca. 745– 750 cm1 and a maximum at ca. 804 nm, which is slightly shifted irradiated with a continuous wave fiber-coupled diode laser source regarding the two characteristic absorption peaks of this PS located at 808 nm (50 W, Oclaro, Inc., San Jose, CA). The used power fluen- at the NIR window at the measured conditions, one at 720 nm (oli- cies were 0.5, 1.0, and 2.0 W/cm2 for 5 min. After 18 h and 42 h, gomeric form) and another at 820 nm (monomers) [47], cells were washed again, and new fresh culture medium (100 lL) respectively. was added with 10 lL of CCK-8 reagent to each well and measured f-potential and FTIR data also confirmed the successful cover- as specified above. Experiments were repeated at least three times. age of AuNRs by the PE layers. A complete surface charge reversal after coating the particles with PSS and PLL-ICG conjugate was 2.12. Statistical analysis observed as consequence of their different electrical charge (Fig. 1c). For HA, we controlled its deposition to obtain slightly pos- Statistical analysis of experimental data was performed with itively charged particles, which should be relatively more effective Origin software. All results were presented as mean standard devi- in being uptaken by negatively charged cells [48], as mentioned ation unless otherwise stated. One-way ANOVA (*P < 0.05; previously. Fig. 1d shows that the FTIR spectrum of PSS/PLL- **P < 0.01) was used to determine statistical differences for multi- coated AuNRs displayed bands at 1236 cm1 belonging to C-N ple groups, whereas unpaired t-test was used to analyzed individ- stretching, at 1545 and 1639 cm1 corresponding to NH2 bending ual groups. and C = O stretching of PSS, 2948 cm1 to asymmetric C–H stretch- ing, and finally, at 3415 cm1 for symmetric NH2 and O–H stretch- 3. Results and discussion ings, respectively. Meanwhile, the FTIR spectrum of free ICG confirmed the presence of characteristic vibrational modes at 3.1. Synthesis and characterization of PSS/PLL-ICG/HA AuNRs 883, 1277, 1409, 2923 and 3460 cm1 corresponding to N–H tor- nanoplatforms sion, C-O, C-N and C = C stretchings, C–H bending and N–H sym- metric stretching, respectively. The presence of ICG within PSS/ Bare AuNRs were prepared by means of a well-established two- PLL-ICG/HA-coated AuNRs was reflected by the development of step seed-mediated growth method with slight modifications [13]. peaks at ca. 1407 and 1280 cm1 corresponding to C-N stretching The obtained anisotropic metallic NPs exhibited a size of ca. and C = C vibrations of the PS aromatic rings. Finally, the HA coat- 33  9 nm with two absorption bands: a weak shorter transversal ing was confirmed by the development of additional peaks at ca. 5 L.G. Arellano, E.M. Villar-Alvarez, B. Velasco et al. Journal of Molecular Liquids 377 (2023) 121511 Fig. 1. (a) UV–vis absorbance spectra of PSS/PLL-ICG/HA-coated AuNRs after each coating deposition step. Some physical properties for the nanoplatform are also shown: b) Longitudinal LSPR shifts (j), absorbance decreases from the longitudinal ( ) and transversal modes ( ); and c) f-potentials. Solid and dashed lines are only to guide the eye. d) FTIR spectra of CTAB-coated AuNRs ( ), free ICG (—), PSS/PLL coated AuNRs ( ) and PSS/PLL-ICG/HA coated AuNRs ( ). TEM images of e) CTAB-coated AuNRs; f) PSS- coated AuNRs; g) PSS/PLL-ICG-coated AuNRs; and h) PSS/PLL-ICG/HA-coated AuNRs. Asterisks in d) indicated the main peaks discussed in the text. 769 cm1 and 545 cm1 assigned to C-O stretching vibrations and observed, respectively, with no further decreases observed at a-glycosidic linkages and O–H extensions, respectively. longer incubation times [50]. Conversely, the photostability of both free ICG and PSS/PLL-ICG/ 3.2. Photo- and chemical stabilities of PSS/PLL-ICG/HA-coated AuNRs HA-coated AuNRs was reduced after their NIR light excitation, and such destabilization was more important as the fluence increased: As commented previously, ICG is a water-soluble tricarbocya- For PSS/PLL-ICG/HA-coated AuNRs the observed fluorescence nine dye that strongly absorbs and emits light in the NIR region reduction was ca. 28%, 25% and 14% after 15 min of NIR illumina- of the electromagnetic spectrum. However, for intended biomedi- tion at 2.0, 1.0 and 0.5 W/cm2, respectively, which was rather sim- cal applications it is necessary to carefully consider the NIR light ilar to that obtained, for example, with other types of anisotropic dose to be applied to maximise ROS and/or heat generation while gold nanoparticles as nanoshells, in which ICG was entrapped precluding dye photodegradation and thermal decomposition as a inside the polymeric core [55]. It was clearly observed that the con- consequence of temperature increases from non-radiative thermal jugation of the dye to the PLL polymer and subsequent integration decays [49–54]. For these reasons, the photostability of free ICG into the nanoplatform surface coating enhanced the photochemical and PSS/PLL-ICG/HA-coated AuNRs was compared for ca. 20 days protection of ICG, as confirmed by comparison with the strong loss in the absence and presence of NIR light illumination. Fig. 2a, of fluorescence signal of the free ICG dye (ca. 44%) after 15 min of shows that the stability of the fluorescence signal from ICG com- NIR laser irradiation at 2.0 W/cm2 (Fig. 2b). In summary, the pre- plexed to AuNRs was enhanced compared to that of free ICG at sent observations evidenced that the dye-PE complex conjugated similar concentration (22 lM, 84% dye encapsulation efficiency to the hybrid nanoplatform was relatively stable upon continuous within the nanoplatform) in the absence of light excitation. An ini- wave (CW) laser illumination at the selected NIR irradiation dura- tial fluorescence intensity reduction of ca. 15% was noted for PSS/ tions and fluencies. PLL-ICG/HA-coated AuNRs after 2 days of incubation, much lower To analyze the protection provided by the AuNR-based platform than 70% detected for the free ICG dye; after one week, additional to the conjugated ICG dye under relevant physiological conditions, reductions of ca. 20 and 15% of the fluorescence signals were ICG release profiles at both neutral and acidic conditions (pH 7.4 6 L.G. Arellano, E.M. Villar-Alvarez, B. Velasco et al. Journal of Molecular Liquids 377 (2023) 121511 Fig. 2. (a) Time evolution of fluorescence intensity of free ICG ( ) and PSS/PLL-ICG/HA-coated AuNRs (d). (b) Photostability of PSS/PLL-ICG/HA-coated AuNRs under continuous illumination with NIR light of 2.0 ( ), 1.0 (d) and ( ) 0.5 W/cm2. Fluorescence intensity of free ICG at the maximum fluence is also shown for comparison ( ). Lines are only to guide the eye. c) ICG cumulative releases from PSS/PLL-ICG/HA coated-AuNRs in the absence ( , ) and presence (j, ) of trypsin at pH 5.0 ( , j) and 7.4 ( , ), respectively. d) ICG cumulative release from PSS/PLL-ICG/HA-coated AuNRs at pH 5.0 in the absence of trypsin and NIR light (j), in the presence of trypsin ( ), in the presence of NIR illumination at 1.0 ( ) and 2.0 ( ) W/cm2, and the simultaneous presence of both trypsin and NIR light irradiation at 1.0 ( ) and 2.0 ( ) W/cm2 provided by a laser source of 808 nm (for 5 min), respectively. e) Zoom of the initial release area showing the stair-like release profile of the dye upon NIR illumination. Lines are only to guide the eye. Arrows denote the time points of irradiations: 2, 5, 9, 12 and 48 h. Measurements were performed in triplicate (n = 3). and 5.0) in the presence of 10% (v/v) FBS were monitored for 96 h massive diffusion of the PS out of the coating layer despite the pro- at 37 °C. Moreover, the influence of both endogenous (the protease gressive degradation of the PLL polymer by trypsin. This was espe- trypsin) and exogenous (NIR light) triggers to induce ICG release cially noted at acidic pH, in which the PLL (pKa  9–10) layer were also considered. In vitro cumulative ICG release profiles at electrostatically interacts with the underlying PSS chains (pKa  both neutral (pH 7.4) and acidic conditions (5.0, mimicking lysoso- 1–2) even in the presence of the enzyme compacting the PE coating mal pH) showed a burst phase followed by a much slower diffusion and, thus, offering protection to avoid excessive dye diffusion out release pattern. of the nanoplatform. The release profiles were slightly faster and larger in the pres- On the other hand, when applying 808 nm CW NIR laser irradi- ence of trypsin, as expected by the protease-induced PLL degrada- ation at 1.0 and 2.0 W/cm2 for 5 min after 2, 5, 9, 12, and 48 h of tion [56], -ca. 26% and 40% at pH 5.0 and 7.4 compared to ca. 20% incubation, ICG release extents at pH 5.0 after 96 h,were ca. 32 and 32% in the absence of the enzyme after 96 h of incubation, and 38%, respectively, values slightly larger than in the presence respectively, see Fig. 2c. However, the final release extents were of the enzyme (Fig. 2d). It is worth mentioning that light-induced low since amide bonding upon ICG conjugation to PLL hinders a releases were characterized by a stair-like profile, that is, a strong 7 L.G. Arellano, E.M. Villar-Alvarez, B. Velasco et al. Journal of Molecular Liquids 377 (2023) 121511 Fig. 3. Temperature increases along incubation under NIR light irradiation (808 nm). Three power intensities were tested: 0.5 W/cm2 (a), 1.0 (b) and 2.0 W/cm2 at two nanoplatform concentrations 11011 NPs/mL (a-c) and 11010 NPs/mL at 1.0 W/cm2 (d) for (j) PSS/PLL-ICG/HA-coated AuNRs, ( ) PSS/PLL/HA-coated AuNRs, ( ) free ICG (equivalent concentration to that loaded inside PSS/PLL-ICG/HA-coated AuNRs), and ( ) pure aqueous solution. Measurements were performed in triplicate (n = 3). burst phase just right after the application of the light external heat conversion in PSS/PLL-ICG/HA-coated AuNRs provided by stimulus followed by a progressive levelling off at longer times. the metallic core and ICG dye in order to elucidate whether there This provides an excellent control over the cargo release kinetics exists a competitive or synergistic effect for light-induced heat on demand. production. The light-triggered temperature increments under Moreover, the combination of both internal (enzyme) and 808 nm NIR light irradiation of PSS/PLL-ICG/HA-coated AuNRs, external (light) stimuli enhanced the release rate compared to PSS/PLL/HA-coated AuNRs, and free ICG were obtained at three the application of individual stimuli, with up to ca. 46 and 59% of laser fluencies (0.5, 1.0, 2.0 W/cm2) and two particle concentra- entrapped ICG being released under 1.0 and 2.0 W/cm2 NIR light tions (11011 and 11010 NP/mL). Free ICG concentrations of 22 irradiation + trypsin at pH 5.0, respectively, which are much higher and 2.2 lM, respectively, similar as those present in AuNR-based if compared with quantities detected after the application of the nanoplatforms, were used as controls. individual NIR light (32 and 38%, respectively) or trypsin triggers Pure aqueous solutions increased their temperature<1 °C under (24% under both conditions). any irradiation condition along the time interval tested. Con- versely, temperature increments provided by free ICG (at 22 lM) 3.3. NIR-induced temperature heat profiles of PSS/PLL-ICG/HA-coated were ca. 1.6, 2.8 and 6.0 0C under 0.5, 1.0 and 2.0 W/cm2, respec- AuNRs tively, which confirms the effective capacity of this dye to convert light into heat [58–61]. For the nanoplatforms, either PSS/PLL/HA- As shown in Fig. 1, the ICG absorption spectrum when the dye is coated or PSS/PLL-ICG/HA-coated AuNRs, particle and fluence bound to the nanoplatform showed a shoulder at ca. 745–750 nm dependences of the reached solution temperatures were clearly and a maximum at ca. 802 nm, as mentioned previously. After light observed. Strong temperature increases of up to 22.6 and 13.8 0C absorption, an ICG molecule can follow three different pathways to were observed for PSS/PLL/HA-coated and PSS/PLL-ICG/HA-coated deactivate its excited state [57]: i) The absorbed energy can be con- AuNRs under irradiation of 2.0 W/cm2 at a particle concentration verted into heat by internal conversion mechanisms (ca. 80–85 %) of 1.01011 NPs/mL, respectively, with a temperature maximum [58]; ii) the absorbed energy can be emitted as fluorescence light after 15 min which does not further change. Similar temperature [32]; iii) part of the absorbed light is transferred to the ICG triplet increments were also obtained for other types of anisotropic T1 state, which enables the generation of ROS when relaxed to the nanoparticles as Au nanoshells [55] or nanoprisms [62]. As com- forbidden singlet state [59]; Therefore, heat generation is also a mented previously, this increment stem from the excitation of major process after light absorption by ICG (extinction coefficient, the dye to their triplet state and subsequent relaxation to the sin- eICG = 1.1104 M1cm1 at 778 nm), and which is added to the heat- glet one, but also to the relaxation of Au metal hot electrons and to-light conversion induced by the metallic core (AuNR) of subsequent energy transfer upon their relaxation to the crystal lat- nanoplatforms (eAuNR = 2.0109 ± 0.1109 M1cm1). Then, it tice of the metallic core, which further relaxes through heat emis- appears necessary to analyze the nature of the coupled light-to- sion [23,44]. When decreasing the particle concentration to 8 L.G. Arellano, E.M. Villar-Alvarez, B. Velasco et al. Journal of Molecular Liquids 377 (2023) 121511 1.01010 NP/mL, the observed temperature increase was rather cessful uptake of the hybrid nanoplatform by HeLa cells after 6 h of lower, ca. 2.4 and 2.1 0C, respectively, and with a maximum incubation was confirmed by both TEM and epifluorescence micro- detected after ca. 10–12 min of irradiation. In addition, by compar- scopy images (Fig. 4e-g and Fig. 5). TEM images confirmed the ing PSS/PLL-ICG/HA-coated AuNRs and their PSS/PLL/HA-coated incorporation of the NPs inside the cell cytoplasm within endo- counterparts lower temperature enhancements were observed somes (Fig. 4e-g), with a perimembrane location. Moreover, light for the former, and these differences were generally larger as the reflected by the metal-core hybrid NPs also denoted that these fluence increased. Such observations might stem from the ability were present inside cells in the form of small aggregates. In addi- of ICG to absorb NIR light, thus, competing with the light-to-heat tion, the in vitro ROS production by PSS/PLL-ICG/HA-coated AuNRs metal NP conversion. In the former case, after light absorption under 808 nm NIR laser illumination of 1.0 W/cm2 was qualita- ICG returns to its ground state through fluorescent radiative decay, tively evaluated at different time intervals by epifluorescence singlet oxygen production and/or by internal conversion (non- microscopy (kex = 490 nm/kem = 520 nm) in cancerous cervical radiative decay) with heat emission, being then a less efficient pho- HeLa cells by means of a fluorometric assay (see Experimental Sec- tothermal agent than the metal NPs. It is also known that absorp- tion for details). Untreated HeLa cells were used as the negative tion cross section of Au NPs is orders of magnitude stronger than control, and cells supplemented with H2O2 (800 lM) as the posi- that of most strongly absorbing molecules [13]. In this case, the tive one. Fig. 5 shows that green fluorescence signal and, hence, molar extinction coefficient of AuNRs obtained was ca. ROS production increased as the irradiation time was longer. e = 2.109 ± 1.109 M1cm1, in agreement with previous data [63]. Untreated cells did not show any fluorescence signal as expected, This value is 5 orders of magnitude larger than the molar extinc- whereas some scarce background signal was also detected when tion coefficient of ICG (e = 1.08  104 M1 cm1 at 778 nm), so that the nanoplatform was internalized into cells in the absence of the energy absorbed by the dye is not converted into heat so effi- NIR illumination due to either some metabolic stress generated ciently as in the case of metal AuNRs cores [64]. Hence, the by uptake (in agreement with toxicity data, see below) and/or observed temperature increments in the presence of the dye the existence of some light reflection from the nanoplatforms within the polymeric coating of Au NRs were lower, [26]; on the themselves. other hand, part of the incident energy can be also transmitted On the other hand, a relative measure of ROS production after 6 to PE layers decreasing the heat conversion efficiency. Despite and 24 h of incubation at fluencies of 1.0 and 2.0 W/cm2 (808 NIR the PEs used herein do not absorb at NIR frequencies hot electrons light) for 5 and 10 min of irradiation at different solution temper- produced by the longitudinal plasmon decay might be transferred atures (4, 25 and 37 °C) was additionally performed using a plate to the wrapped PEs [65]. reader. The temperature dependent production of ROS was here considered in order to decouple the potential contributions from 3.4. In vitro singlet oxygen generation photodynamic and photothermal responses to cell cytotoxicity (see below for details). Untreated and H2O2-treated cells were used The possibility of using the PSS/PLL-ICG/HA-coated AuNR as the negative and positive controls, respectively. An additional nanoplatform as a potential PDT agent was tested by measuring control consisting of PSS/PLL/HA-coated AuNRs was also its 1O2 generation capability under NIR laser irradiation at considered. 808 nm. The generation of 1O2 was quantitatively determined by Fig. 6a shows that the fluorescence signal denoting the presence following the absorbance intensity decrease of the DPIBF probe of ROS was enhanced at a fluence of 2.0 W/cm2 with ca. 78–80%, at 420 nm, which is a specific singlet oxygen trap, at different time much larger that 57–59% obtained under to 1.0 W/cm2 after intervals [66]. 5 min of irradiation, compared to the positive H2O2 control 1 O2 production was observed to increase (DPIBF absorbance (100%). No significant effects of the macroscopic solution temper- decreases) after 30 min of continuous laser irradiation (Fig. 4d), ature on ROS generation were observed. ROS production was also which certainly indicated that an effective reduction in the 1O2 more important as the irradiation time was extended up to production took place. Also, this was enhanced as the irradiating 10 min (Fig. 6b), with values of ca. 69–71% and 82–84% at 1.0 energy input did (Fig. 4a-d). Interestingly, the 1O2 generation by and 2.0 W/cm2, respectively. Finally, it is clearly observed that PSS/PLL-ICG/HA-coated AuNRs was slightly lower than that of free ROS generation decreased with incubation time as expected, being ICG at similar concentrations since the effective laser energy avail- a consequence of both cell death (see below) upon light exposition able for 1O2 production was lower in the former case. For the and the progressive relaxation of the excited dye to the ground nanohybrid platform, the laser energy was simultaneously state, thus, reducing ROS generation. absorbed by the dye and the metal core to produce both singlet oxygen species and heat, and the accessibility of the dye to the 3.6. In vitro therapeutic activity in tumoral cells oxygen dissolved in the aqueous solution medium was also more reduced since the PS is inside the polymeric coating [15,67,68]. It The therapeutic activity of the PSS/PLL-ICG/HA-coated AuNR is also worth mentioning that pure aqueous solution and PSS/ platform was assessed by means of the CCK-8 proliferation assay PLL/HA-coated AuNRs also led to certain transformation of DIPBF, at three different temperatures, 4, 25, and 37 °C at different NIR as noted by slight decreases in its absorbance. This effect stemmed fluencies (1.0 and 2.0 W/cm2), incubation (6 and 24 h) and irradi- from the nature of the primary oxidation product of DPIBF, an ation times (5 and 10 min) in cervical cancerous HeLa cells in order unstable ozonide which cleaves into a dibenzoylbenzene plus an to identify the origin of the cytotoxic response of the platform unknown oxygenated product under continuous NIR light irradia- under light excitation and the existence or not of potential thera- tion in aqueous environment [69–71]. Both samples exhibited the peutic synergistic/additive effects under dual PDT/PPTT bi-therapy. same absorbance reductions since they did not truly generate 1O2 Fig. 7a shows that under 1.0 W/cm2 NIR light irradiation for by themselves, so they can be used as references of the DPIBF 5 min, the PSS/PLL/HA-coated AuNR platform hardly provides a degradation process. photothermal therapeutic effect except at 37 °C, for which cell tox- icity reaches ca. 18%; for this solution temperature, the thermal 3.5. In vitro ROS assay in tumoral cells increment (ca. 2–3 0C, see Fig. 3) is enough to achieve certain cell sensitization leading to the beginning of apoptosis, as previously The capacity of PSS/PLL-ICG/HA-coated AuNRs as potential reported [10,72]. Such an effect is enhanced when the incubation intracellular PDT agent was next evaluated. As a first step, the suc- time is extended up to 24 h, reaching ca. 35% cell death. 9 L.G. Arellano, E.M. Villar-Alvarez, B. Velasco et al. Journal of Molecular Liquids 377 (2023) 121511 Fig. 4. Quantification of 1O2 production through the DPIBF absorbance decrease at 420 nm in pure aqueous solution ( ), or in thre presence of PSS/PLL/HA-coated AuNRs ( ), free ICG free ( ) and PSS/PLL-ICG/HA-coated AuNRs ( ) at (a) 2.0, (b) 1.0, and (c) 0.5 W/cm2. (d) Comparison of 1O2 production for PSS/PLL-ICG/HA-coated AuNRs illuminated for 30 min at 2.0 ( ), 1.0 ( ), and 0.5 ( ) W/cm2. e) TEM image of PSS/PLL-ICG/HA-coated AuNRs internalized within an endosome of a HeLa cell; f) zoomed TEM image; and g) confocal image of PSS/PLL-ICG/HA-coated AuNRs in reflection mode inside HeLa cells. Nuclei are stained with DAPI. Scale bars 1 lm. In (a-d), measurements were performed in triplicate (n = 3). Conversely, when incorporating the ICG dye within the hybrid addition of H2O2 to cells and used as control was very large and nanostructure (PSS/PLL-ICG/HA-coated AuNRs), cell toxicity within 64–68%. increases up to ca. 29–30% in the temperature range 4–25 0C and When increasing the irradiation fluence to 2.0 W/cm2 for 5 min, to ca. 35% at 37 0C after 6 h of incubation, with further increments an additional enhancement of cell toxicity promoted by the PSS/ to 35% and ca. 43% after 24 h of incubation, respectively. These data PLL/HA-coated AuNR platform was observed, which is more rele- confirm then that the encapsulation of ICG promoted the genera- vant after 24 h of incubation and at the highest incubation temper- tion of ROS species under NIR illumination, as confirmed by the ature tested (37 0C, see Fig. 7b). At 4 and 25 0C ROS production and, observed enhancement in cell toxicity at 4 and 25 °C for PSS/PLL- thus PDT, seemed the main contribution to cell death, with cell ICG/HA-coated AuNRs compared to PSS/PLL/HA-coated ones. Here, toxicities lying within the range of 40–44% and 49–54% for PSS/ it is necessary also to remind that the macroscopic temperature PLL-ICG/HA-coated AuNRs after 6 and 24 h of incubation, respec- increments provided by the PSS/PLL-ICG/HA-coated AuNRs are tively. Conversely, cell mortalities were ca. 23 and 50% and 42 lower than those of PSS/PLL/HA-coated ones, so the observed tox- and 61 % for PSS/PLL/HA-coated and PSS/PLL-ICG/HA-coated AuNRs icity enhancement cannot be from thermal origin. On the other after 6 and 24 h of incubation, respectively, at 37 °C. Then, it seems hand, free ICG dye provided cell mortalities between 30 and 35% that as the fluence increases the contribution of the photothermal and 45–50% after 6 and 24 h of incubation, respectively, rather effect to cell death became stronger at the largest incubation tem- similar as those of the PSS/PLL-ICG/HA-coated AuNR platform. It perature by allowing to reach a stronger sensitization or even the is also worth mentioning that toxicity by ROS generated from the cell ablation regime. In this regard, similar observations were also 10 L.G. Arellano, E.M. Villar-Alvarez, B. Velasco et al. Journal of Molecular Liquids 377 (2023) 121511 Fig. 5. Fluorescence microscopy images of ROS production by PSS/PLL-ICG/HA-coated AuNRs within HeLa cells under NIR irradiation with a 808 nm CW laser source (1.0 W/ cm2) for 15 min. Untreated cells were used as a negative control, whereas H2O2-treated cells were the positive one. Direct transmitted light (left), fluorescent green channel (middle, kem = 520 nm for detection kit) and merged images (right) are shown. Scale bars are 75 lm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) noted when using, for example, gold nanoshells with ICG illumination of the nanoconstruct after 10 min at 2.0 W/cm2 (NIR entrapped in their polymeric core, for which cell toxicities of ca. light of 671 nm) were from 15 to 95% in the dye concentration 70 and 80% were observed by combination of PPTT + PDT in the range from 0.25 to 4 lM [67]. nanosystem in HeLa and MDA-MB-231 cancer cells under 2.0 W/ Finally, further enhancements of cell toxicities were observed cm2 for 5 min [55]; in addition, Au nanostars encapsulated in when increasing the irradiation time up to 10 min under illumina- CaCO3 and functionalized with ICG were irradiated for 6 min at tion of 2.0 W/cm2 (Fig. 7c). For PSS/PLL/HA-coated AuNRs, cell mor- 1.0 W/cm2, reaching cell toxicities of up to 92% at a particle con- talities increased from ca. 7, 15 and 23% at 6 h to 47, 50 and 55% centration of 30 lg/mL [73]. In a similar approach, Au nanostars after 24 h of incubation at 4, 25 and 37 0C, respectively, whereas were functionalized with polyethylene glycol-5 kDa (PEG-5 kDa) for PSS/PLL-ICG/HA-coated ones toxicities changed from ca. 3, 16, and incorporated chlorin e-6, Ce6. Cell mortalities induced by the and 39% to ca. 58, 60 and 71% after 6 and 24 h of incubation at 11 L.G. Arellano, E.M. Villar-Alvarez, B. Velasco et al. Journal of Molecular Liquids 377 (2023) 121511 Fig. 6. ROS production induced by PSS/PLL-ICG/HA-coated AuNRs internalised within HeLa cells under NIR irradiation of 1.0 and 2.0 W/cm2 with a 808 nm CW laser source for a period of (a) 5 min and (b) 10 min. Color bars denote the following conditions: 4 °C, 6 h of incubation; 25 °C, 6 h; 37 °C, 6 h; 4 °C, 24 h;, 25 °C, 24 h; 37 °C, 24 h. Untreated cells were used as a negative control whereas H2O2-treated cells were the positive one. Fig. 7. Cell viabilities of HeLa cells after administration of PSS/PLL-ICG/HA-coated AuNRs and subsequent irradiation at a) 1.0 W/cm2 for 5 min; b) 2.0 W/cm2 for 5 min; and c) 2.0 W/cm2 for 10 min at different temperatures and incubation times. Free and encapsulated ICG concentration was 22 lM whereas that of H2O2 was 0.8 mM (*P < 0.05; **P < 0.01; ns = not statistically significant). 12 L.G. Arellano, E.M. Villar-Alvarez, B. Velasco et al. Journal of Molecular Liquids 377 (2023) 121511 Table 1 ablation regimes are achieved leading to either apoptosis or necro- Relationship between external conditions and phototherapy-dominant cytotoxicity. sis thermal-induced cell death pathways, respectively, which are Solution Temperature added to the predominant PDT activity provided by the ICG dye. Fluence /Wcm 2 4 °C 25 °C 37 °C In this manner, we were able to decouple the contributions of PDT and PPTT to cell cytotoxicity as a function of solution temper- 1.0 (t = 5 min) PDT PDT PDT 2.0 (t = 5 min) PDT PDT PDT + PTT ature and irradiation conditions, which are many times obscure or 2.0 (t = 10 min) PDT + PTT PTT PTT directly not performed in similar studies, achieving a clear picture of how this nanoplatform can be more effective for its intended therapeutic application. the same solution temperatures, respectively. Thus, a larger expo- sure time to light irradiation seemed to enhance the photothermal Funding effect whereas ROS generation seemed to be rather maintained after comparing the experimental data corresponding to PSS/PLL/ This research was funded by Agencia Estatal de Investigación HA-coated and PSS/PLL-ICG/HA-coated AuNRs. This behavior might (AEI), project number PID2019-109517RB-I00 and Xunta de Galicia arise from a lower contact of the dye with surrounding water ED431C 2022/18. ERDF funds are also acknowledged. A.C. also molecules, which is key to generate 1O2 and OH– species, after its thanks Xunta de Galicia for grant number ED481D-2021–011. L. encapsulation within the polymeric coating. Direct contact of free A. is grateful to CONACYT for funding her PhD fellowship. ICG dye with water should facilitate both ROS generation and heat diffusion to the surroundings being reflected, in general, in slightly larger cell toxicities except at 37 0C after 24 h of incubation. Table 1 Data Availability Statement shows a brief summary indicating the main cytotoxic photother- apy as a function of the applied external conditions (fluence, irra- The data presented in this study are available on request from diation time, temperature). the corresponding authors. Declaration of Competing Interest. The authors declare that they have no known competing finan- 4. Conclusions cial interests or personal relationships that could have appeared to influence the work reported in this paper. In this work, AuNRs were functionalized through the layer-by- layer technique to provide them with a biocompatible surface layer CRediT authorship contribution statement in which the NIR dye ICG was incorporated by means of its conju- gation with the cationic biopolymer (PLL) used in the coating pro- Lilia G. Arellano: Methodology, Validation, Formal analysis, cess. The obtained hybrid nanoplatform, to give PSS/PLL-ICG/HA- Investigation, Data curation. Eva M. Villar-Alvarez: Methodology, coated AuNRs, was able to provide a dual simultaneous PDT and Validation, Investigation. Brenda Velasco: Methodology, Valida- PPTT therapy for tumor treatment thanks to the presence of ICG tion. Vicente Domínguez-Arca: Software, Visualization. Gerardo and the optical properties of AuNRs, which are favored by both Prieto: Writing – review & editing, Project administration. Adriana the enhanced photostability of the dye and its location to large Cambón: Conceptualization, Data curation, Writing – original extents into the nanoconstruct even after extensive irradiation in draft, Supervision, Funding acquisition. Silvia Barbosa: Conceptu- contrast to previously developed nanosystems for similar pur- alization, Resources, Writing – original draft, Writing – review & poses. In this regard, the dye release rates and profiles were editing, Supervision, Funding acquisition. Pablo Taboada: Concep- observed to be perfectly controlled using both internal (e.g., tualization, Resources, Writing – review & editing, Project adminis- enzymes as trypsin thanks to the degradability of PLL) or external tration, Funding acquisition. (e.g., NIR light thanks to the optical properties of the dye itself and AuNRs) stimuli, if required. Surprisingly, the light to heat conver- sion provided by the PSS/PLL-ICG/HA-coated AuNRs nanoplatform Data availability was smaller compared to that of PSS/PLL/HA-coated AuNRs, that is, the platform without encapsulated dye. This behavior might orig- Data will be made available on request. inate from both the lower absorption cross section of the PS com- pared to the metallic core and competitive light absorption effects. Declaration of Competing Interest The hybrid platform was also able to produce ROS species effi- ciently in test tube and intracellularly, and such generation The authors declare that they have no known competing finan- depended on the fluence and time irradiation but not on the solu- cial interests or personal relationships that could have appeared tion temperature [24,26,27]. Nevertheless, the protection of the to influence the work reported in this paper. dye inside the particle polymeric coating might hinder a more direct contact with oxygen-containing solvent, which may involve References a slightly lower ROS production compared to the free dye at similar concentration and irradiation conditions. It was finally shown that [1] X. Huang, M.A. El-Sayed, Plasmonic photo-thermal therapy (PPTT), Alexandria the application of NIR light, particularly at 2.0 W/cm2 and 37 °C, J. Medicine 47 (2011) 1–9, https://doi.org/10.1016/j.ajme.2011.01.001. [2] H.S. Han, K.Y. 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